Pcdna3.1 hygromycin vector

The human kidney epithelial 293T and BHK-21 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂. The HN gene of NDV (Mukteswar strain; Gene bank: JF950509.1) was synthesized by the GENEWIZ Biotechnology Co., Ltd (Suzhou, China) and subcloned into the transient mammalian expression vector pcDNA3.1 using HindIII and BamHI restriction enzyme sites (Invitrogen). For protein expression, a cDNA fragment encoding the globular head domain of wild-type or mutant HN (residues 147–571), was cloned into a modified pcDNA3.1+hygromycin vector (Invitrogen) containing secretion signal and removable C-terminal Fc tag by thrombin cleavage site.

An oligo-dT–primed cDNA library was constructed in a phage λ ZAP II vector (Agilent Technologies) using poly(A)⁺ RNA from A. punctulata testis. Approximately 500,000 independent recombinants were screened with a PCR fragment encoding a portion of the KHD of the GC (Singh et al., 1988 (link)). A full-length clone of ∼4,000 bp was completely sequenced on both strands. The largest open reading frame codes for a protein of 1,122 amino acid residues with a calculated molecular mass of 126 kD. The sequence was analyzed with PROSITE for potential sites of posttranslational modification. For GC expression, the sequence has been cloned into the pcDNA3.1(+)-hygromycin vector (Invitrogen). To ensure proper membrane targeting, the GC signal peptide (amino acids 1–24) has been replaced with the signal peptide of the human epidermal growth factor receptor (MRPSGTAGAALLALLAALCPA).